Sequential chemical peptide and oligonucleotide syntheses are well established, widely used procedures for producing peptides and oligonucleotides, such as those up to about 40 residues (peptides) and up to 100 residues (oligonucleotides). For peptides, the chemistry involves the specific coupling of the amino terminus of a carboxyl-blocked peptide to the activated carboxyl group of an amino-blocked amino acid. For oligonucleotides, the chemistry involves the specific coupling of the 5'-hydroxyl group of a 3'-blocked nucleotide to an activated 3'-hydroxy group of a 5'-blocked nucleotide.
In their most commonly used forms, developed primarily by Merrifield, J. Amer. Chem. Soc., 85, 2149 (1963) and BeauCage, S. L. and Caruthers, M. H., Tet. Lett. 22, 1859-1862 (1981); Beaucage, S. L. and Caruthers, M. H., J. Amer. Chem. Soc., 24, 3184-3191 (1981), these syntheses are accomplished with the peptide or oligonucleotide immobilized on a solid support. An extremely large number of peptides or oligonucleotides can be produced by this methodology. The physical and chemical properties of the peptide or oligonuoleotide products will vary greatly depending on size and composition of the respective amino acids or nucleotides composing these products. Consequently, it is typical to tailor the synthetic techniques to fit the specific product at hand.
In the method of immobilized peptide synthesis, the carboxyl terminal amino acid is bound to a polyvinyl benzene or other suitable insoluble resin. The second amino acid to be added possesses blocking groups on its amino moiety and any side chain reactive groups so that only its carboxyl moiety can react. This carboxyl group is activated with a carbodiimide or other activating agent and then allowed to couple to the immobilized amino acid. After removal of the amino blocking group, the cycle is repeated for each amino acid in the sequence.
The efficiency of the peptide coupling step usually varies from 95-99.9%, depending on the identity of the amino acid and its location in the sequence. During each coupling step, a small portion of the peptides fail to couple the next amino acid. Since these failures occur independently during each coupling step, the amount of correctly sequenced peptide in the final mixture is often less than a major portion. Failed peptides with incorrect sequences (by virtue of amino acid deletions) often accumulate to a significant degree in this mixture.
The same is true of oligonucleotide syntheses. In general, the oligonucleotide synthetic procedure follows the well-established 3'-phosphoramidite schemes devised by Caruthers The 3' terminal base of the desired oligonucleotide is immobilized on an insoluble carrier. The nucleotide base to be added is blocked at the 5' hydroxyl and activated at the 3' hydroxyl so as to cause coupling with the immobilized nucleotide base. Deblocking of the new immobilized nucleotide compound and repetition of the cycle will produce the desired final oligonucleotide.
As is true for the peptides, this nucleotide coupling procedure is not 100% efficient. The immobilized oligonucleotide molecules that do not couple result in oligonucleotides of incorrect sequences. These often cause undesirable reactions if left in mixture with the correct oligonucleotide. Consequently, their separation and removal are mandated even though tedious procedures tailored to each specific synthesis are necessitated.
Separation of the various peptides or oligonucleotides in the respective mixtures produced during synthesis will produce the desired pure, correctly sequenced peptide or oligonucleotide. Conventional separation techniques usually employ high resolution chromatographic procedures such as reverse phase high pressure liquid chromatography, electrophoresis, gel chromatography and the like. These separation method(s) need to resolve peptides or oligonucleotides which differ from each other by as little as one amino acid or nucleotide. The failed peptides and oligonucleotides are compounds having physical and chemical properties very similar to the desired one. Consequently, the separations are difficult to accomplish. Since the compounds synthesized can vary greatly in composition, the monomeric unit sequence and length, the separation methods also are individually tailored to the properties of each mixture. Such separation procedures are difficult to develop, require many man-hours to implement and do not insure absolute homogeneity of the product.
One means for attacking this problem involves increasing the coupling yield. This can be accomplished by performing repeated couplings at each coupling step prior to the next deblocking step. But repeated couplings provide only a partial solution to producing pure peptides or oligonucleotides. The repeated coupling steps expend larger quantities of expensive agents and protected amino acids or nucleotides. In manual synthesis, the coupling yield is monitored at each step before deciding whether to repeat the coupling step, whereas automated synthesis is severely restricted in this respect. Moreover, some peptides or oligonucleotides fail to couple completely during the chain elongation because the large size of the activated amino acid or nucleotide prevents access to some of the peptides or oligonucleotide molecules on the resin. Therefore, these methods are severely limited in scope.
Another means for attacking this problem involves "capping". This method reduces the total number of incorrectly sequenced or "failed" peptides or oligonucleotides in the synthetic mixture. To cap, the failed peptides or oligonucleotides are reacted with a capping agent which prevents the failed peptide or oligonucleotide from participating in subsequent coupling reactions (for peptides, see Merrifield, J. Amer. Chem. Soc. (1963) 2149; Markley and Dorman, Tetrahedron Letters (1970), 1787; for oligonucleotides, see Efimov, V.A., Chakhmakhcheva, O. G., and Ovchimikov, V.A. Nucleic Acids Res. 13, 3651 (1985)).
As applied to peptides, capping can be accomplished because the extended (i.e., coupled) peptide possesses a blocked amino group at the N-terminus while the failed peptides possess a free N-terminus amino group. Once the failed peptide is capped, it is unavailable for further coupling steps. The result is a mixture of capped failed peptides of different lengths and the correctly extended peptide without a cap.
As applied to oligonucleotides, capping can be accomplished because the failed oligonucleotide contains a free 5'-hydroxyl group. Capping with an irreversible agent that reacts with hydroxyl groups will prevent further reaction of this failed side product. The cap will not react in any subsequent steps of the oligonucleotide synthetic procedure.
A modification of the capping strategy employs a capping agent which changes the chemical or physical properties of the failed peptides or oligonucleotides (Penke and Birr Justus Liebigs Ann. Chem., 1999 (1974), and Krieger et al., Proc Nat. Acad. Sci. 3160 (1976)). Such modifications augment the chemical and physical differences between the correctly extended peptide or oligonucleotide and the failed peptides or oligonucleotides. These differences tend to aid separation.
Nevertheless, these capping methods for reducing the contamination of synthetically produced peptides or oligonucleotides have drawbacks. No matter what the capping agent, the overall physical characteristics of the peptides or oligonucleotides usually determine their physical and chemical behavior. The resulting separations remain dependent upon the overall physical and chemical behavior of the peptides or oligonucleotides. Very tedious and time consuming separations result because the overall properties of the desired product and side products are much the same.
Therefore, it is an object of the invention to develop a synthetic method for the preparation of peptides and oligonucleotides that yields pure product. Another object is to develop a method that avoids time-consuming separation techniques. Yet another object is to base this method upon a capping technique.